Oncotarget, 2017, Vol. 8, (No. 25), pp: 40606-40619 Research Paper The G-protein coupled receptor 56, expressed in colonic stem and cancer cells, binds progastrin to promote proliferation and carcinogenesis Guangchun Jin1,2, Kosuke Sakitani1, Hongshan Wang1,3, Ying Jin1, Alexander Dubeykovskiy1, Daniel L. Worthley4, Yagnesh Tailor1 and Timothy C. Wang1 1Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA 2The Research Institute, Yanbian University Hospital, Jilin, China 3Department of General surgery, Zhongshan Hospital, Fudan University, Shanghai, China 4South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, South Australia, Australia Correspondence to: Timothy C. Wang, email: [email protected] Keywords: GPR56, progastrin, proliferation, stem cell, colorectal cancer Abbreviations: Human progastrin (hGAS), G-protein coupled receptor 56 (GPR56), 5-bromo-2’-deoxyuridine (BrdU), Bone morphogenetic protein (BMP). Received: November 02, 2016 Accepted: February 22, 2017 Published: March 23, 2017 Copyright: Jin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56 mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis. INTRODUCTION aberrant glands that progress pathologically from aberrant crypt foci into small adenomatous polyps and ultimately Colorectal cancer (CRC) is the third most commonly invasive adenocarcinoma. Hyperproliferation of colonic diagnosed cancer in the world [1]. CRC develops through mucosal epithelial cells has been found to be a critical a series of somatic gene mutations that promote malignant initiating event in colorectal carcinogenesis [3]. Growth behavior [2]. These traditional pathway mutations, factors and hormones can modulate colonic mucosal propagated by long-lived progenitor cells, lead to activation epithelial cells proliferation and thus an individual’s underlying predisposition to colorectal cancer. www.impactjournals.com/oncotarget 40606 Oncotarget Preprogastrin is synthesized by gastric G cells, and stem cells during brain development [23]. GPR56 has also cleavage of the signal peptide at the C-terminus leads to the been shown by Irving Weissman’s group to be expressed in production of progastrin [4]. Progastrin can undergo further hematopoietic stem cells [24]. Taken together, these data processing to generate glycine-extended and amidated raise the possibility that GPR56 may function to control the forms of gastrin. While progastrin and other nonamidated proliferation or behavior of multipotent stem cells of diverse gastrins typically comprise less than 10% of the total origins. GPR56 does not appear to be required for survival secreted peptide, elevated levels have been described in of adult mammals since knockout mice are viable [25]. some gastrointestinal cancer patients [4, 5], and progastrin Although GPR56 may also interact with tissue collagen III has been shown to be expressed by numerous primary human tumors and cancer cell lines [6–8]. Amidated gastrins were initially thought to be the only biologically “orphan receptor” with unknown functions. active form of gastrin. However, in recent years, progastrin In addition, GPR56 is overexpressed in numerous has been recognized as a growth factor. Overexpression cancers, including glioblastomas, breast, pancreatic, renal, of human progastrin in transgenic mice, or progastrin esophageal cancers, and colon cancer [20, 28–30]. In treatment in vitro, stimulates colonic epithelial proliferation and colonic carcinogenesis, proving that progastrin is a observed in transformed cells compared with its isogenic trophic growth factor for colonic epithelium in mice [9–12]. nontransformed revertant, and GPR56 silencing by RNAi Furthermore, overexpression or exogenous administration approaches led to growth suppression in vitro and tumor of progastrin increases colonic epithelial proliferation and regression in xenograft tumor models in vivo [28]. A regeneration after DNA damage by gamma-radiation or smaller number studies have pointed to a possible role for chemical carcinogens [13]. These effects of progastrin were GPR56 as a tumor suppressor gene as it is downregulated independent of other forms of amidated gastrin [14]. Recent studies have suggested that progastrin’s effects in cancer. GPR56 has been shown to interact effects on colonic mucosal proliferation and carcinogenesis with both Gaq/11 and Gq12/13, and activate a number are mediated through inhibition of bone morphogenetic of downstream signaling pathways including ERKs, protein (BMP) signaling [11]. Overall, while proliferative NF-kB, cAMP, and most importantly Wnt signaling [31, and protumorigenic effects of progastrin have been 32]. Studies by Shashidhar et al have shown that GPR56 demonstrated both in vivo and in vitro, the receptors and overexpression results in the upregulation of TCF reporter pathways that mediate progastrin induced proliferation genes, implicating the beta-catenin pathway in GPR56 signaling [30]. binding proteins of progastrin have been reported by other In this study, we demonstrated that progastrin groups [11, 15–18]. Recent reports by our group have binds to GPR56- expressing colon cancer cells, and demonstrated that the cholecystokinin type 2 receptor utilizing GPR56-CreER™ transgenic mice, that GPR56 (CCK2R) when expressed in colon cancer cells can bind to is expressed in a subset of stem cells in the colonic crypt. progastrin, while antagonism or knockout of the CCK2R Deletion of GPR56 abrogates progastrin-dependent receptor in vivo abrogates progastrin’s proliferative and protumorigenic effects [11]. CCK2R is expressed, carcinogenesis in mice. Although a few GPCRs have been however, only at very low levels in the normal murine considered as potential cancer drug targets, our studies colon, thus raising the question as to whether CCK2R is suggest that GPR56 plays an important role in mediating indeed the primary receptor mediating progastrin’s effects. the effects of progastrin induce colonic proliferation and As an orphan G protein–coupled receptor (GPCR), colon carcinogenesis and thus could serve as a valuable GPR56, is a member of the class secretin-like GPCR future target to prevent and treat colorectal carcinogenesis. subfamily with an extremely long extracellular domain thought to play a role in cell-cell and cell-matrix interactions RESULTS [19]. GPR56 is highly expressed in the brain, thyroid gland and heart, with moderate levels in kidney and pancreas, GPR56 is expressed in murine colonic crypt cells small intestine, stomach, and colon [19, 20]. In the brain, and upregulated in human progastrin transgenic GPR56 is expressed in the germinal zones of fetal and mice adult brain regions harboring neural stem cells, and there is a strong link between GPR56 and stem cell function While GPR56 is widely expressed in murine across a wide range of distinct compartments. For instance, neuronal, muscle, and thyroid cells [19, 33], the expression of GPR56 in the gastrointestinal epithelium has not been while overexpression increases proliferation and progenitor number in neuron [21]. Mutations in GPR56 have been linked to bilateral frontoparietal polymicogyria [22], which was higher in the stomach than in the small intestine and is due to altered migration and proliferation of neuronal colon in 6-week-old WT C57BL/6 mice (Figure 1A). www.impactjournals.com/oncotarget 40607 Oncotarget Additionally, in situ hybridization of GPR56 (Figure 1B) hGAS mice leads to increases in GPR56-expressing cells, particularly in the setting of carcinogenic injury. 1C) detected GPR56 positive epithelial cells located near the base of the colonic crypts. In addition, more numerous GPR56-expressing cells could be detected in progastrin- cells overexpressing hGAS/GPR56-EGFP mice compared to the WT/GPR56-EGFP